Apparatuses and methods for modulating fluids using acoustically oscillating solid structures

ABSTRACT

An acoustofluidic apparatus is provided that functions by the generation of microstreaming in fluid produced by the oscillation of oscillatory elements excited by an energy input such as acoustic energy. An acoustofluidic apparatus includes an oscillatory energy field generator in energetic contact with one or more oscillatory elements contained in a fluid passage. Oscillation of the oscillatory elements induces microstreaming in a fluid that can be used to mix laminar flows of differing fluids, as a micropump for the directional movement of fluid through a fluid passage, for the generation of waveforms in a fluid or plurality of fluids or for other purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application depends from and claims priority to U.S. PatentApplication No: 61/884,907 filed Jul. 11, 2013, the entire contents ofwhich are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No.ECCS0824183, awarded by the National Science Foundation and Grant No.OD007209 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

Modulation of fluid flow in a microfluidic channel is demonstrated viathe acoustic microstreaming phenomenon induced by the oscillation ofoscillatory elements. By optimizing the design of the oscillatoryelements, excellent fluid modulation performance can be achieved in asimple device, making the acustofluidic apparatus a promising candidatefor a wide variety of applications.

BACKGROUND OF THE INVENTION

The ability to achieve rapid and homogeneous mixing ofchemical/biological species enables a wide variety of applications, suchas chemical kinetic studies^(1,2) and nanomaterial synthesis.³⁻⁷ Whilemicrofluidic devices seem to be an excellent platform for carrying outthese studies due to their short reaction times, high throughput, andreduced reagent consumption, effectively mixing fluids at the microscaleis not a trivial process.⁸⁻¹⁶ Due to inherently small channeldimensions, the flow of fluid in microfluidic devices is usuallylaminar. Under laminar flow conditions viscous forces dominate overinertial forces and fluids are not easily mixed. In order to enablemicrofluidic applications that require mixing, a number of micromixingmethods have been reported. These methods include: chaoticadvection,¹⁷⁻²¹ hydrodynamic focusing,²²⁻²⁵ electrokinetically drivenmixing,²⁶⁻³¹ 3D combinatorial mixing,^(32,33) meandering channels aswell as magnetically,^(34,35) thermally,³⁶ and optically³⁷ inducedmixing.

Recently, acoustic-based micromixers have attracted significantattention due to their non-invasive nature³⁸⁻⁴² and simple mixingmechanism. In acoustic-based mixers, acoustic waves propagate into fluidmedia and induce pressure fluctuations, resulting in the disturbance ofthe laminar-flow pattern to facilitate mixing.⁴³⁻⁴⁹ The mixingperformance of acoustic-based mixers can be further improved through theuse of bubbles in the microfluidic channel. When bubbles are coupledwith an acoustic wave, the acoustic streaming phenomenon⁵⁰ is developed.This phenomenon results in a more prominent perturbation of thesurrounding fluids, greatly facilitating the mass transport of fluids.Thus far, bubble-based acoustic mixers⁵¹⁻⁵⁴ have been used forcharacterizing enzyme reactions,² enhancing DNA hybridization,^(51,55)generating chemical gradients,⁵⁶ and developing advanced optofluidicdevices.⁵⁷ Although acoustically driven, bubble-based micromixers haveshown tremendous potential in a wide variety of applications, there aremany concerns regarding bubble instability,^(53,57) heat generation,⁴⁸and inconvenient bubble-trapping processes. To take advantage ofacoustic streaming without the drawbacks of microbubbles,⁵⁸⁻⁶¹ there isa need to explore alternative methods that can effectively andconveniently generate acoustic micromixing.

In addition to mixing of fluids on the microscale level, significantefforts have been made towards developing reliable, robust microfluidicpumps. Prior pumps have been characterized as either active or passive.Passive pumps such as surface-tension based microfluidic pumps offerseveral advantages including their simple operation, low cost andsemi-automation; however, they are vulnerable to evaporation caused byenvironmental changes, and to a flow rate changing with time. Activepumps that use mechanical or electrical systems to initiate fluidpumping may potentially provide solutions to the challenges that passivepumps encounter. Active pumps offer the advantages of flexibility interms of temporal control of pumping behaviour and adjustable flowrates. Most prior active pump systems require sophisticated fabricationprocesses for patterning electrodes and for making multi-layer devices,complicated optical setups, or well-trained personnel to operate thedevices, limiting active pumps from being developed and integrated as an“on-chip” pumping unit for handheld, portable platforms.

Overall, there is a great need for mechanisms that can be used formodulating fluids on the microscale level that are robust, simple tomanufacture and operate, and have excellent flexibility for a broadrange of applications such as inducing mixing, generating concentrationor temporal gradient profiles, generating fluid flow such as in apumping apparatus, producing activity in rapid on/off format forusefulness in waveform control, and other possible uses.

SUMMARY OF THE INVENTION

The following summary of the invention is provided to facilitate anunderstanding of some of the innovative features unique to the presentinvention and is not intended to be a full description. A fullappreciation of the various aspects of the invention can be gained bytaking the entire specification, claims, drawings, and abstract as awhole.

Novel apparatuses and methods are provided to control a chemicalmicroenvironment using oscillatory elements capable of oscillating in anacoustic field where the oscillation is useful to produce arbitrarytemporal waveforms in a flow system such as buffer solutions. Examplesof the invention include using an acoustically activated, oscillatoryelement based microfluidic system for generating arbitrary temporalchemical waveforms (both digital and analog) by mixing the stimuli andbuffer solutions in a time-dependent fashion. This approach permitscontinuous modulation of the signal characteristics including shape,frequency, amplitude, and duty cycle, with frequencies reaching up to 30Hz, and in some examples frequencies greater than 30 Hz.

By incorporating oscillatory structures in ladder-like, parabolic, orother arrangements into a single fluid channel, both static andpulsatile chemical gradients are achievable. With its advantages infunctionality and versatility, the chemical waveform generation andswitching methods presented herein are powerful tools that may be usedin many biological and chemical applications.

As such, it is a first object of the invention to provide an apparatususeful for inducing microstreaming in a fluid. An apparatus is providedthat is useful for many such processes including a fluid passage, saidfluid passage comprising at least one sidewall, at least one inlet, andat least one outlet; an oscillatory element comprising a tip extendinginto said fluid passage; and an acoustic source in acoustic contact withsaid oscillatory element, said acoustic source operable to vibrate saidoscillatory element or a portion thereof, so as to create microstreamingin a fluid within said fluid passage. The oscillatory element optionallyincludes two adjacent surfaces extending between said sidewall and saidtip, said adjacent surfaces being disposed at a tip angle with respectto each other, the tip angle being less than 180 degrees, optionallyless than or equal to 45 degrees, optionally less than or equal to 30degrees, optionally less than or equal to 15 degrees. I someembodiments, the surface is not in the form or a point, but instead hasa surface with a width and a length, the length is optionally but notrequired to be equal to the height of said fluid channel. Optionally,the width is from 5 to 100 micrometers. In some embodiments, theoscillatory element is extending at a tilting angle relative to thelength of said sidewall, said tilting angle being 90 degrees or less,optionally any angle in either direction from 0 degrees to 70 degrees.In some embodiments, the apparatus includes a plurality of oscillatoryelements with two or more of said oscillatory elements having differentoscillating frequencies. An acoustic source is optionally apiezoelectric transducer. In some embodiments, an acoustic source isoperable to vibrate the oscillatory element at a frequency in the rangeof 2 Hz to 900 MHz, optionally at least 1 kHz. It is appreciated thatany of the claimed elements can be combined in ways other thanexplicitly recited herein.

It is another object of the invention to provided methods of inducingmicrostreaming in a fluid. Provided are processes of inducingmicrostreaming in a fluid including contacting a first fluid in a fluidpassage with a oscillatory element; and acoustically oscillating saidoscillatory element with an acoustic source in acoustic contact withsaid oscillatory element so as to induce microstreaming in said firstfluid. In some embodiments, the oscillatory element comprises a tipangle of 45 degrees or less. Optionally, the oscillatory element isoriented relative to an edge of said fluid passage by a tilting anglefrom 0 degrees to 70 degrees. Optionally, the oscillatory elementoscillates at a frequency in the range of 2 Hz to 900 MHz, optionally 1kHz, optionally more than 1 kHz. In some embodiments, the microstreaminginduces mixing of said first fluid and a second fluid, and optionally athird fluid, within said fluid channel. In some embodiments, themicrostreaming induces flow of the fluid in the fluid channel by themicrostreaming being directionally oriented. Optionally, a processinduces both pumping (e.g. fluid flow) and mixing of a first fluid and asecond fluid. The process optionally produces a concentration gradientof a component of said first fluid, said second fluid, or both.Optionally, the process further includes contacting said first fluid andsaid second fluid with a plurality of oscillatory elements off set andgenerating a gradient profile between said first fluid and said secondfluid. Optionally, the process further includes producing aconcentration gradient of a component of said first fluid, said secondfluid, optionally said third fluid, or any combination thereof. Theplurality of oscillatory elements are optionally arranged in an angledladder configuration, a parabolic configuration, or a V form. A processoptionally includes alternating said acoustic source from an on state toan off state to produce a temporal gradient waveform of concentration ofsaid first fluid and said second fluid. As such, the microstreaming isoptionally operable to produce fine or gross gradients of concentrationbetween two or more fluids in a flow passage that is useful for manyassays such as cellular or other bio assays, binding of particularelement, or many other uses. Fluid can be simultaneously mixed andpumped in a channel, or either mixed or pumped at one or otherlocations. Some embodiments induce mixing. Some embodiments, inducingpumping of fluid. Some embodiments induce both mixing and pumping. Rapidcontrol over on/off status of the acoustic source allows both temporaland special control of element concentrations in a region of interestallowing generation of chemical waveforms that can be used for manytypes of studies.

It is another object of the invention to provide processes for pumping afluid through a channel, optionally a microchannel, including contactinga fluid in a fluid passage with an oscillatory element, said oscillatoryelement, or oscillating portion thereof, oriented relative to an edge ofsaid fluid passage by a tilting angle from 0 degrees to 180 degrees; andacoustically oscillating said oscillatory element with an acousticsource in acoustic contact with said oscillatory element so as to inducedirectional microstreaming in said fluid. In the processes, theoscillatory element(s) are optionally at a tilting angle of 0 degrees to70 degrees in either direction relative to the edge of a flow passage.Optionally, the oscillatory element oscillates at a frequency in therange of 2 Hz to 900 MHz, optionally at least 1 kHz. Pumping is inducedby the oscillation of the fluid passages. Pumping is optionally tightlycontrolled in time and space by controlling the off/on status of theacoustic source so as to be capable of generating temporal waveforms offluid flow at desired locations in a flow passage. The pumping action isoptionally used with a mixing action between two or more fluidscontaining different chemical (optionally biological) components.

Overall, the apparatuses and processes provided address many issues withprior devices including simple and low cost manufacture, tightregulation of activity, rapid on/off responses, and the ability togenerate fine chemical gradient profiles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an acoustofluidic apparatus according to oneembodiment;

FIG. 1B is a schematic of exemplary microstreaming produced byoscillation of a oscillatory element acoustofluidic apparatus accordingto one embodiment;

FIG. 1C illustrates the dimensions and spacing of a fluid passage andoscillatory elements in an acoustofluidic apparatus according to oneembodiment;

FIG. 1D illustrates the dimensions and spacing of a fluid passage andoscillatory elements arranged at a tilting angle in an acoustofluidicapparatus configured as a micropump according to one embodiment;

FIG. 2A illustrates laminar flow of fluid in a fluid passage when anexemplary acustofluidic device is not activated;

FIG. 2A illustrates laminar flow of fluid in a fluid passage when anacustofluidic device is activated to cause oscillation of theoscillatory elements in the fluid passage producing microstreaming ofthe fluid about the oscillatory elements;

FIG. 3A illustrates laminar flow of two adjacent fluids introduced bytwo separate inlets into a fluid passage when an exemplary acustofluidicdevice is not activated with the dashed line depicting a region ofinterest for the quantification of mixing of the two flows;

FIG. 3B illustrates incomplete mixing of two adjacent fluids introducedby two separate inlets into a fluid passage when an exemplaryacustofluidic device is activated with an acoustic frequency of 4.25kHz;

FIG. 3C illustrates complete mixing of two adjacent fluids introduced bytwo separate inlets into a fluid passage when an exemplary acustofluidicdevice is activated with an acoustic frequency of 4.5 kHz;

FIG. 3D illustrates complete mixing of two adjacent fluids introduced bytwo separate inlets into a fluid passage when an exemplary acustofluidicdevice is activated with an acoustic frequency of 4.75 kHz;

FIG. 3E illustrates the normalized dye concentration profile across theflow passage width for the three driving frequencies of 4.25 kHz, 4.50kHz, and 4.75 kHz;

FIG. 4A illustrates an acoustofluidic apparatus according to oneembodiment used to quantitatively characterize the mixing performancealong the entire length of the flow passage with 5 regions of interestindicated as 1, 2, 3, 4, and 5;

FIG. 4B illustrates mixing with a tip angle of 15 degrees demonstratingcomplete mixing by position 2;

FIG. 4C illustrates mixing with a tip angle of 30 degrees demonstratingcomplete mixing by position 5;

FIG. 4D illustrates mixing with a tip angle of 45 degrees;

FIG. 4E illustrates no observable mixing with a tip angle of 60 degrees;

FIG. 4F illustrates the mixing performance of an exemplaryacoustofluidic apparatus dependent on tip angle;

FIG. 5A illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees oscillated by a transducer with a driving voltage of 15V;

FIG. 5B illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees oscillated by a transducer with a driving voltage of 23V;

FIG. 5C illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees oscillated by a transducer with a driving voltage of 31 V;

FIG. 5D illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees oscillated by a transducer with a driving voltage of 39V;

FIG. 5E illustrates the mixing performance of an exemplaryacoustofluidic apparatus dependent on driving voltage;

FIG. 6A illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees and a flow rate of 1 μl/min with a driving frequency of4.50 kHz and a driving voltage of 31 Vpp;

FIG. 6B illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees and a flow rate of 2 μl/min with a driving frequency of4.50 kHz and a driving voltage of 31 Vpp;

FIG. 6C illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees and a flow rate of 3 μl/min with a driving frequency of4.50 kHz and a driving voltage of 31 Vpp;

FIG. 6D illustrates mixing in an acoustofluidic device with a tip angleof 15 degrees and a flow rate of 4 μl/min with a driving frequency of4.50 kHz and a driving voltage of 31 Vpp;

FIG. 6E illustrates the mixing performance of an exemplaryacoustofluidic apparatus dependent on fluid flow rate;

FIG. 7A is a schematic of an exemplary acoustofluidic device and tworegions of interest (ROI) as used for determining calcium responses ofU251 cells due to differing concentrations of agonist as generated byoscillation of the oscillatory elements;

FIG. 7B illustrates laminar flow of two fluids when the device of FIG.7A is inactive;

FIG. 7C illustrates mixing of the two fluids when the device of FIG. 7Ais activated generating an intermediate concentration of agonist at ROI1and ROI2.

FIG. 7D illustrates temporal control over mixing achieving differentchemical signal profiles at two the ROIs;

FIG. 8A illustrates normalized concentration of agonist at the two ROIsunder exemplary temporal control;

FIG. 8B illustrates normalized concentration of agonist at the two ROIsunder exemplary temporal control;

FIG. 8C illustrates normalized concentration of agonist at the two ROIsunder exemplary temporal control;

FIG. 8D illustrates normalized concentration of agonist at the two ROIsunder exemplary temporal control;

FIG. 9A illustrates an acoustofluidic apparatus for fine gradientproduction according to one embodiment;

FIG. 9B illustrates an acoustofluidic apparatus for fine gradientproduction according to one embodiment;

FIG. 10A illustrates side-by-side laminar flow of the fluids in theembodiment of FIG. 9A when the apparatus is not activated;

FIG. 10B illustrates mixing of fluids when the apparatus of FIG. 9A isactivated at 23 Vpp;

FIG. 10C illustrates mixing of fluids when the apparatus of FIG. 9A isactivated at 31 Vpp;

FIG. 10D illustrates mixing of fluids when the apparatus of FIG. 9A isactivated at 39 Vpp;

FIG. 10E illustrates the gradient profiles achieved transversely acrossthe fluid passage at various voltages;

FIG. 11A illustrates side-by-side laminar flow of the fluids in theembodiment of FIG. 9B when the apparatus is not activated;

FIG. 11B illustrates mixing of fluids when the apparatus of FIG. 9B isactivated at 23 Vpp;

FIG. 11C illustrates mixing of fluids when the apparatus of FIG. 9B isactivated at 31 Vpp;

FIG. 11D illustrates mixing of fluids when the apparatus of FIG. 9B isactivated at 39 Vpp;

FIG. 11E illustrates the gradient profiles achieved transversely acrossthe fluid passage at various voltages;

FIG. 12A illustrates a schematic of an acustofluidic micropump accordingto one embodiment with oscillatory elements arranged at a tilting angleaccording to one embodiment;

FIG. 12B illustrates exemplary directional microstreaming achieved as aresult of activation of oscillatory elements at a tilting angle lessthan 90 degrees;

FIG. 12C illustrates exemplary dimensions of elements in the pumpingregion of a micropump according to one embodiment;

FIG. 13A illustrates an exemplary micropump with a fluid passagedesigned to produce fluid pumping in a counter-clockwise directionemploying 30° tilted oscillatory elements as shown in greater detail inthe inset;

FIG. 13B illustrates microstreaming patterns around the tips ofoscillatory elements in the pumping region when the piezoelectrictransducer was activated at 6.5 kHz;

FIG. 14A illustrates the pumping performance of the four differenttilting angles of oscillatory elements under different input voltagesusing the apparatus of FIG. 12A; and

FIG. 14B illustrates alternately pulsatile fluid pumping achieved byswitching the piezoelectric transducer to ON and OFF at various burstfrequencies, 0.5 Hz (T=2 sec) and 2 Hz (T=0.5 sec) using the apparatusof FIG. 12A.

DETAILED DESCRIPTION OF THE INVENTION

The following description of particular embodiment(s) is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly. While the processes or compositions are described as an order ofindividual steps or using specific materials, it is appreciated thatsteps or materials may be interchangeable such that the description ofthe invention may include multiple parts or steps arranged in many waysas is readily appreciated by one of skill in the art.

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. In contrast, when an element isreferred to as being “directly on” another element, there are nointervening elements present.

It will be understood that, although the terms “first,” “second,”“third” etc. may be used herein to describe various elements,components, regions, layers, and/or sections, these elements,components, regions, layers, and/or sections should not be limited bythese terms. These terms are only used to distinguish one element,component, region, layer, or section from another element, component,region, layer, or section. Thus, “a first element,” “component,”“region,” “layer,” or “section” discussed below could be termed a second(or other) element, component, region, layer, or section withoutdeparting from the teachings herein.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms, including “at least one,” unless the content clearly indicatesotherwise. “Or” means “and/or.” As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. It will be further understood that the terms “comprises”and/or “comprising,” or “includes” and/or “including” when used in thisspecification, specify the presence of stated features, regions,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof. The term “or a combination thereof” means a combinationincluding at least one of the foregoing elements.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

Provided are acustofluidic apparatuses that can be configured for manypurposes including mixing of fluids, actively pumping fluid flow,generating chemical/biochemical gradient, creating tuneable chemicaldistribution, or many other uses. Embodiments include methods andapparatuses using one or more oscillatory elements driven by an energyfield (optionally acoustic) to provide a unique and versatile method togenerate prescribed temporal chemical gradient waveforms by mixing twoor more fluids, such as first and second liquid flows (e.g. stimulus andbuffer solutions), optionally in a time-dependent manner, or to movefluid through a channel. This approach is capable of generating not onlydigital chemical waveforms, but also analog waveforms whosecharacteristics, including shape, frequency, amplitude, and duty cycle,can be modulated by controlling the oscillation of one or moreoscillatory elements within a flow channel

In some embodiments, an apparatus is in the form of a micromixeroperable to intermix fluids that addresses issues with prior micromixersin that the micromixer does not require bubbles for operation andeffectively causes tuneable mixing of a fluid or other materialcontained within a fluid passage. The apparatus functions by theincorporation of one or more oscillatory elements that terminate in orotherwise include a tip that will oscillate when in acoustic contactwith one or more acoustic sources so as to produce microstreaming influid or other material.

As such, an acustofluidic apparatus is operable for generating achemical gradient in a fluid flow, or for actively pumping a fluidicchemical material within a fluid passage. As used herein the word“chemical” is understood to include both chemical and biological such asin the case of cells or other multichemical living or non-living system.An apparatus includes a fluid passage, the fluid passage including atleast one inlet. Optionally a fluid passage includes a first inletconfigured to introduce a first fluid flow into the fluid passage, and asecond inlet configured to introduce a second fluid flow into the flowchannel. The fluid passage also includes at least one outlet where oneor more fluids in either a mixed or non-mixed state may be dischargedfrom the fluid passage. An apparatus includes one or more oscillatoryelements that each include a tip extending into the fluid passage. Incommunication with the oscillatory element is an oscillatory energyfield generator, optionally an acoustic source, the generator operableto produce oscillation in the oscillatory element. The oscillation ofthe oscillatory element induces microstreaming in one or more fluidflows optionally generating a chemical gradient having a time-dependencecontrollable using the oscillatory energy field generator. The result isa mixing at the chemical level of the one or more fluids where theparameters of the mixing are controlled or controllable by the energyfield produced by the energy field generator. The result of theapparatus is the ability to tightly control all parameters of mixing orpumping of one or more fluids within the flow channel thereby allowingthe creation of gradients tuned for a desired concentration, time, orarea in the flow.

An acoustofluidic apparatus includes: a channel defining a fluidpassage, the channel having at least one sidewall, at least one inletand at least one outlet; an oscillatory element comprising a tipextending into the fluid passage; and an acoustic source in acousticcontact with the oscillatory element, the acoustic source operable tovibrate the oscillatory element so as to create microstreaming in afluid within said fluid passage.

A fluid passage is a portion of or an entire channel capable ofcontaining a fluid, optionally a flowing fluid. A fluid passage isoptionally formed of transparent or opaque material. In manyembodiments, a fluid passage is located on a substrate. The substrate isoptionally formed of the same of different material as the fluidpassage. The fluid passage is optionally formed from a solid material. Afluid passage is optionally formed from polymer such aspolydimethylsiloxane (PDMS), polypropylene (PP), polyethyleneterephthalate (PET), polybutylene terephthalate (PBT), polycarbonates(PC), polyethylene (PE), polylactic acid (PLA), nylon, PET copolymers,acrylics, Surlyn™, polyethylene naphthalate (PEN), polyamides,polycarbonate co-polymers, elastomeric polymers—thermoplasticelastomers, thermoplastic urethanes, poly urethanes, acrylicco-polymers, acrylonitrile butadiene styrene, or other thermoplastics,glass such as borosilicate glass or other glass material, quartz, steeloptionally stainless steel, gold, combinations thereof, or othermaterial known in the art and suitable for such a purpose. A fluidpassage optionally has a surface roughness that is sufficiently smoothto allow laminar flow of the fluid moving within the flow channel.

The fluid passage(s) optionally has a cross-sectional shape that iscircular, oval, rectangular, square, trapezoidal, triangular, irregular,or other shape. Optionally, the shape of the fluid passage varies withlinear distance along the flow direction, or intended flow direction, ofthe fluid. A fluid passage has length longitudinal to the fluid flowthat is optionally linear or generally linear, curved, angled, irregularor other desired shape. An exemplary cross-sectional dimension of afluid passage is in the range of 1 μm to 30 mm or greater, or any valueor range therebetween. A cross-sectional dimension of a fluid passage isoptionally 1 μm to 10 mm, optionally 1 μm to 1 mm, optionally 10 μm to 1mm, optionally 100 μm to 500 μm. The cross-sectional dimension isoptionally configured to correspond to the type of fluid passing throughthe fluid passage taking into account considerations of viscosity,chemical or biological content, or other necessary parameters.

A fluid passage includes one or more inlets and one or more outlets. Aninlet represents an opening through which a fluid may pass to enter thefluid passage or portion thereof. An outlet is an opening through whicha fluid may pass to exit the fluid passage or portion thereof. In asimplified, non-limiting embodiment, two inlets are present and oneoutlet is present. Typically, the number of inlets corresponds to thenumber of differing fluids to pass into the fluid passage duringoperation of the apparatus. In some embodiments, the outlet is of largercross sectional dimension that an inlet or other portion of the flowchannel.

A fluid passage is optionally a microchannel. A microchannel is a fluidpassage with a cross-sectional dimension on the order of micrometers orless. A fluid passage optionally has one side or edge defined by thesubstrate material. A fluid passage optionally has a width and lengthparallel to the plane of a substrate. A fluid passage also has a heightthat extends in a direction perpendicular (i.e. normal) to a substrate.The height of a fluid passage is optionally from 1 μm to 10 mm orgreater. A height of a fluid passage is optionally 1 μm to 1 mm,optionally 5 μm to 1 mm, optionally 10 μm to 1 mm, optionally 100 μm to500 μm. A width of a fluid passage in a direction parallel to asubstrate surface or perpendicular to a fluid flow direction is anywidth suitable for containing the number of fluids to be flowed throughthe passage.

A fluid passage contains a fluid during operation of the apparatus.Optionally, a fluid passage surrounds a fluid. A fluid is optionally aliquid at testing temperatures and pressures. A fluid is optionally abiologically compatible media such as water or buffered liquidillustratively including phosphate, tris(hydroxymethyl)aminomethane(tris), citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), or other buffering system. A fluid is optionally water, saline,an organic liquid, or other desired flowable material. A fluid isoptionally a gel. A fluid is optionally a suspension of one or moretypes of suspended particles, cells or other substance. A fluidoptionally contains one or more test substances. A test substance is anychemical or biological material that is desired for testing. A fluid hasa test substance concentration. Optionally, a first fluid and a secondfluid contain the same or different test substances or concentrationsdepending on the desired outcome of the system. Optionally, a firstfluid and a second fluid are different types of fluids illustrativelybut not limited to an organic and an aqueous fluid respectively, or viceversa. The fluid type in many embodiments is non-limiting other than thefluid is capable of moving through the flow channel.

A fluid passage is optionally presented on a substrate either by thesubstrate being adjacent to the fluid passage or integrated with thefluid passage as an edge or wall portion. A substrate is any material ofsuitable shape and dimension to support a fluid passage, and optionallyany structure located within the fluid passage. A substrate isoptionally suitable to conduct or transfer energy from an oscillatoryenergy field generator so as to transfer the energy to the oscillatingstructure thereby providing the desired oscillation of the oscillatingstructure. A substrate is optionally made form a polymeric material,illustratively polypropylene (PP), polyethylene terephthalate (PET),polybutylene terephthalate (PBT), polycarbonates (PC), polyethylene(PE), polylactic acid (PLA), nylon, PET copolymers, acrylics, Surlyn™,polyethylene naphthalate (PEN), polyamides, polycarbonate co-polymers,elastomeric polymers—thermoplastic elastomers, thermoplastic urethanes,poly urethanes, acrylic co-polymers, acrylonitrile butadiene styrene, orother thermoplastics, glass such as borosilicate glass or other glassmaterial, quartz, steel optionally stainless steel, silicon, aluminum,gold, combinations thereof, or other material known in the art andsuitable for such a purpose.

A fluid passage includes one or more oscillatory elements. Anoscillatory element includes a tip extending into the fluid passage. Insome embodiments, the entire oscillatory element is located within thefluid passage such that laminar flow around the oscillatory element mayoccur in the absence of oscillation of the oscillatory element(s). Theoverall structure of the oscillatory element includes at least a portionof the oscillatory element that has an oscillating structure whereby theoscillating structure is operable to oscillate at an oscillatoryfrequency when contacted by acoustic or other energy capable of inducingoscillation in the oscillatory element. An oscillatory element isoptionally in the form of a sharp edge, a narrow edge or point ispresent at the tip of the oscillatory element. The tip extends into thefluid passage relative to a wall or other surface of the fluid passage.Optionally, the tip extends from the substrate to the opposing surfaceof the fluid passage, or near enough thereto so as to substantiallyproduce microstreaming in the fluid in the height of the fluid passage.Optionally, an oscillatory element is substantially in the form of a rodor a needle.

A fluid passage optionally includes a single oscillatory element,optionally two oscillatory elements. The number of oscillatory elementsis limited only by the length of the fluid passage, the dimensions ofthe oscillatory element(s), and the desired use of the apparatus.Optionally, a fluid passage includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more oscillatory elements. Insome embodiments, oscillatory elements are dispersed on a single wall ofa fluid passage. Optionally, oscillatory elements are dispersed on twoopposing sides of a fluid passage. When oscillatory elements are presenton opposing sides, the arrangement of the oscillatory elements on afirst edge is optionally offset or directly across from oscillatoryelements on the opposing or second edge. A plurality of oscillatoryelements is optionally uniformly distributed, randomly distributed, orcombinations thereof. Optionally, oscillatory elements serving differentpurposes are dispersed within other oscillatory elements servingdisparate purposes. For example, in an exemplary embodiment, a pluralityof oscillatory elements suitable for pumping fluid are arranged upstreamfrom oscillatory elements suitable for producing fluid intermixing orother purpose. In an alternate exemplary embodiment, a plurality ofoscillatory elements suitable for pumping fluid are arrangedinterspersed with oscillatory elements suitable for producing fluidintermixing or other purpose.

Oscillatory elements are optionally dispersed at a distance relative toan adjacent oscillatory element. A distance is optionally 5 mm or less,optionally 3 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm,200 μm, or less. In some embodiments, the distance of the oscillatoryelement is 30% to 500% the width of the fluid passage. In someembodiments, the distance is equal to the fluid passage width.

An oscillatory element is of suitable composition and configuration soas to be capable of oscillating, or a portion thereof capable ofoscillating, when exposed to energy from an acoustic source, as oneexample. An oscillatory element is or includes a structure that iscapable of oscillation, but does not need to be oscillating at alltimes. Oscillation is defined as movement about a central parameter suchas movement side to side or other direction, by movement due to flexingof an outer dimension of an oscillating structure, or by otherrecognized oscillatory movement.

An oscillatory element terminates in a tip oriented on a position of theoscillatory element distal from a base associated with a surface of afluid passage. A tip is optionally in the form of a sharp angle definedby the terminus of two adjacent surfaces oriented at an angle relativeto each other. Adjacent surfaces extend between a sidewall or othersurface of a fluid passage and the tip. The angle of the adjacentsurfaces a is optionally less than 180 degrees. The angle α isoptionally less than or equal to 60 degrees, optionally less than orequal to 30 degrees, optionally less than or equal to 15 degrees.

An oscillatory element is oriented relative to the longitudinaldirection of the fluid passage in the region of the oscillatory elementat a tilting angle (β). The tilting angle is optionally used to generatemicrostreaming that is directionally oriented, optionally to cause achange in fluid flow. The change in fluid flow is optionally used topump the fluid in a desired direction within the fluid passage. Atilting angle is optionally from 0 degrees to 180 degrees or any valueor range therebetween. Optionally, a tilting angle is 0 degrees, 30degrees, optionally 45 degrees, optionally 60 degrees, optionally 70degrees, optionally 90 degrees, optionally 140 degrees.

An oscillatory element has a length and a width. A length is optionally5 mm or less, optionally 500 μm or less, optionally 400 μm or less,optionally 300 μm or less, optionally 200 μm or less. The length of anoscillatory element is governed by the width of the fluid passage suchthat the length cannot be too long so as to substantially impede fluidflow through the channel. An oscillatory element optionally has a lengththat is less than half the width of a flow channel, but it isappreciated that the oscillatory element is optionally longer than halfthe width of the flow channel optionally in the case where theoscillatory element is not oriented with a length that is normal to thesurface from which it extends.

An oscillatory element has a width. The width of an oscillatory elementdefined as substantially perpendicular to the length of the oscillatoryelement, where the width is optionally uniform or variable along thelength of the oscillatory element. When an oscillatory element is in theform of a sharp edge, a portion of the oscillatory element is wider atthe portion near the base of the oscillatory element relative to thewidth near the tip.

In some embodiments the adjacent edges are parallel or terminate in atip prior to the adjacent edges forming an angle such that the tip is ablunt surface relative to an angle. A blunt surface has a width. Thewidth is optionally from 1 μm to 5 mm or any value or rangetherebetween.

An apparatus includes an oscillatory energy field generator operable toproduce oscillation in the oscillatory element(s). An oscillatory energyfield generator is any device capable of producing energy that willimpart oscillation in an oscillating particle. Energy is optionallyacoustic, electrical, optical, magnetic, or other energy. Devicescapable of generating such energies are known in the art. The exemplaryembodiments herein describe an acoustic source that creates acousticenergy that is felt or received by the oscillatory element causingoscillation in the oscillatory element or portion thereof when theenergy is of the correct parameters to produce such oscillation. It isappreciated that acoustic energy and acoustic energy generators arepresented herein for exemplary purposes alone and not as a limitation onthe present invention. An oscillatory energy field generator isoptionally a piezoelectric transducer. Acoustic-based oscillatingstructure manipulation methods are excellent alternatives toconventional methods. Compared to their optical, electrical, or magneticcounterparts, acoustic-based methods are relatively non-invasive tobiological objects and work for most microparticles regardless of theiroptical, electrical, or magnetic properties.

An oscillatory energy field generator is optionally a chirp interdigitaltransducer (IDT) or other acoustic energy generating device. Anoscillatory energy field generator is formed or attached to thesubstrate and when energized by an input signal creates a vibration inthe substrate. This vibration passes into the oscillating structuredirectly or indirectly via an intermediate structure to produceoscillation in the oscillatory element(s). An electronic control circuitis optionally wired to the oscillatory energy field generator to producethe input signal thereby producing the energy field. This circuit maytake a variety of forms as is known in the art.

The energy field produces an oscillation of the oscillatory element orportion thereof that is in physical contact with one or more fluids inthe flow channel (when present). The oscillation of the oscillatingstructure induces a mixing of the fluid by creating microstreaming inthe fluid. The resulting microstreaming optionally generates a chemicalgradient between two or more fluids, induces a pumping action on afluid, or combinations thereof. A chemical gradient or pumping actionhas a time-dependence, spatial dependence, concentration dependence, orcomposition dependence controllable by the oscillatory energy fieldgenerator.

An apparatus can be manufactured by several processes useful for formingmicrochannel apparatuses. Illustrative examples include, but are notlimited to laser cutting, waterjet cutting, injection molding,photolithography, soft lithography, chemical etching, CNCmicromachining, vacuum molding, stamping, among others, or combinationsthereof. Illustrative methods such as photolithography andmicromachining are commonly used in the semiconductor manufacturing artsare useful for forming an acustofluidic apparatus. In some embodiments,individual elements are separately manufactures and bonded together toform an apparatus using bonding materials as recognized in the art.

An exemplary embodiment of an acoustofluidic apparatus is illustrated inFIG. 1A depicting an experimental setup of a sharp-edge-basedacoustofluidic micromixer 10. A single-layer fluid passage 12,optionally formed from polydimethylsiloxane (PDMS), has eightoscillatory elements illustrated as a sharp edge 14 on its sidewalls 16(four edges on each side). The illustrated embodiment is fabricated andbonded onto a glass slide 18. An acoustic source illustrated as apiezoelectric transducer 20 (model no. 273-073, RadioShack Corp.) isthen attached adjacent to the fluid channel 12 by using epoxy(PermaPoxy™ 5 Minute General Purpose, Permatex). Upon the actuation ofthe piezoelectric transducer, the sharp-edges 14 acoustically oscillateto generate a pair of counter-rotating vortices 22, 24 (double-ringrecirculating flows, micromixing) in the fluid around the tip of eachsharp-edge 14, as shown in FIG. 1B. The double-ring recirculating flowsdrastically enhance the mass transport across the channel width bybreaking the interface of laminar fluids and optionally causing a mixingbetween two or more laminar fluid flows.

While the sharp edges are illustrated as pointed elements having atriangular shape with two flat surfaces, other shapes may be used foralternative embodiments. In one example, oscillatory elements take theform of thin rectangular elements, having generally parallel sidesurfaces and a short end surface. This results in two “tip angles” of 90degrees. Further alternatives include structures with two side surfacesthat curve or angle in near the outer end to define a pointed tip. Thismay have a shape similar to a sharpened pencil as viewed in a side view.Further alternatives may also be used, as long as sufficient acousticstreaming results, allowing acceptable levels of mixing.

The illustrated structures are very small and are two dimensional. Thatis, they have a constant cross section in the z-dimension. The channelhas a top to bottom dimension (perpendicular to the page of the drawing)and the illustrated structures extend from the top to the bottom with auniform shape. Alternatively, three dimensional shapes may be used, suchas a cone, post or needle that extends into the fluid passage. Othershapes will be clear to those of skill in the art.

Though not illustrated, the height of the oscillatory elements may bevaried. In the illustrated embodiment, the oscillatory elements extendfrom each side wall and are interdigitated. The combined height of theoscillatory elements on opposite sides is 500 μm (250 μm each) while thechannel has a total width of 600 μm. Put another way, the channel may besaid to have only 100 μm of clear space. As such, the oscillatoryelements extend more than 80% of the width of the channel (more than 40%from each side). The height may be varied depending on the desiredperformance characteristics. Also, in some embodiments, oscillatoryelements may extend from only a single side. However, it is preferredthat the oscillatory elements extend across a total of at least 10% ofthe channel and that oscillatory elements extend from opposed sides ofthe channel to provide this combined total. Oscillatory elements, suchas extending 25% or more across the channel may be used in someembodiments. Greater than 75% may be used in some applications.

FIG. 1C shows an exemplary design of an acustofluidic micromixer withsidewall sharp-edges serving as an oscillating structure. The length,width and depth of the illustrated microchannel are 1 cm, 600 μm, and 50μm, respectively. Each sharp-edge oscillatory element is designed to beof a constant height of 250 μm and tip angle (α), optionally variable.The sharp-edge may also be referred to as an oscillating structureoptionally serving as an oscillatory element or pumping element. In theillustrated embodiment, the oscillatory element is a pointed elementhaving two surfaces 30 and 32 extending away from the pointed tip 34,with the surfaces 30 and 32 defining the tip angle α therebetween. Whilethe surfaces 30 and 32 are illustrated as flat, other shapes arepossible that are regular or irregular. While sharp-edges are attachedto the sidewall in the illustrated embodiment, they should also workwhen they are in other parts of the channel. Five different tip angles(180° illustrating a blunt end surface, 15°, 30°, 45° and 60°) werechosen to investigate the resulting acoustic streaming effect anddetermine the optimal angle for best mixing performance.

FIG. 1D schematically shows an exemplary design and working mechanism ofan acoustofluidic apparatus functional as an acoustofluidic pump. Theacoustofluidic pump, briefly, is made by bonding a single-layer PDMSfluid passage 16 with a piezoelectric transducer (Part no.81-7BB-27-4L0, Murata Electronics) attached adjacent to it onto a singleglass slide using a thin layer of epoxy (PermaPoxy™ 5 Minute GeneralPurpose, Permatex). The pumping region illustrated in FIG. 1D, isdesigned with twenty tilted oscillatory elements 14 on the sidewalls ofthe fluid passage (ten on each side). Acoustically oscillated by theactivation of the piezoelectric transducer, the tilted oscillatoryelements generate a tilted micromixing pattern around the tip 34,thereby producing a net force pointing towards the direction thatoscillatory element structure is orientated. As a result, fluid pumpingoccurs because the generated net forces push the bulk fluid to flowforward. In this exemplary design the microchannel has a width of 600 μmand a depth of 100 μm, and oscillatory elements are identical. Differenttilting angles (β) of oscillatory elements, including 30°, 45°, 60° and70°, are created to investigate the resulting pumping behaviour anddetermine optimal angle for best pumping performance.

Applications of the apparatus include microfluidic devices (as usedhere, this term includes nanofluidic devices), for chemical, biological(including molecular biology, cell migration, cytotoxicity), andbiochemical (including enzyme, protein, DNA, RNA, proteomics, pathology,and the like) analysis, assay, detection, modification, interaction,preparation, treatment, or characterization applications. Applicationsalso include a fluid mixing apparatus for any application, includingchemical formulations, inkjet apparatus, chemical deposition, filmformation, and the like, optofluidic devices (e.g. to obtain gradientrefractive indices, for example for lens arrays), and the like. Specificapplications include nanofluidic devices, chemical probing of cells, andprogrammable chemical waveform generation and switching usingacoustically activated bubbles. Examples include apparatuses and methodsfor generating chemical concentration or physical (e.g. electricaland/or optical property) gradients that may be dynamically controlled byan electronic circuit, e.g. one providing a variable drive signal to apiezoelectric transducer.

Fluid flows are optionally liquids, suspensions, and the like. Flows mayinclude suspended particles, such as biological structuresillustratively including, but not limited to cells, platelets, orproteins, among others. Applications include characterization ofparticles such as cells, including cell chemotaxis, celldifferentiation, and cell migration studies in a dynamic chemicalenvironment.

Hence, spatial and temporal chemical gradient profiles are achievedusing one or more acoustically driven oscillating oscillatory elementslocated within a fluid passage, for example using a single oscillatoryelement located within the fluid passage, or a plurality of oscillatoryelements, for example positioned in a ladder-like formation within theflow channel. Changing the applied voltage of a drive signal applied toan acoustic transducer such as a piezoelectric transducer dynamicallytunes the generated chemical gradient profiles, both spatially andtemporally. More complex and abundant chemical profiles through changinglocation(s) of the oscillatory element, for example, may be made. Thedesign of the ladder-like formation may be modified using differentconfigurations of the oscillatory elements within the flow channel.Chemical gradients may be adjusted using flow rate control of inletfluid flows in combination with drive signal modification.

Acoustofluidic-based methods and apparatus for generating chemicalgradient can be used in many chemical and biological studies andapplications, such as apparatuses and methods for investigating cellchemotaxis, differentiation, and migration in a dynamic chemicalenvironment.

Using many embodiments of the apparatus, it is possible to measure thedynamics of receptor-mediated signaling and other cellular responses tosmall molecules. The device can also be used to study cellular processesthat span a wide range of time scales, from milliseconds to hours.Generating waveforms in continuous flow also eliminates the abruptchanges in shear stress at the cell membrane in segmented flow devices,more closely mimicking the in-vivo chemical signals. These preciselycontrolled chemical waveforms can be used for measuring the kinetics offast enzymatic reactions, explaining the specificity and efficiency ofgene expression, and developing time-release drugs, among otherapplications. Chemical waveforms may have markedly different effects oncellular signaling pathways that receive, transmit, process, andimplement directions from chemical stimuli, compared with constantsignals, and arbitrary chemical waveforms can be determined.

Examples of the present invention further include apparatuses andmethods for generating tuneable, pulsatile chemical gradient generationvia acoustically driven oscillating bubbles.

A novel concept of generating both static and pulsatile chemicalgradients using acoustically activated oscillatory elements wasdeveloped, in some examples using a ladder-like arrangement. Theseresults show that the chemical gradient profiles can be effectivelytuned by regulating the amplitude of the oscillatory elementoscillation.

Pulsatile chemical gradients generated in microfluidic devices may beused for the characterization of dynamic biological and chemicalprocesses. Spatial and temporal characteristics of chemical stimuli playan important role in cell signalling, and hence this may be investigatedusing described approaches.

Pulsatile chemical gradients may also be used in an apparatus andmethods for high-throughput characterization of cellular processes suchas directed migration, differentiation, and apoptosis. Apparatuses andmethods allow dynamic temporal control of chemical gradients to beachieved.

Also provided are processes of inducing microstreaming in a fluidincluding contacting a fluid in a fluid passage with an oscillatoryelement and oscillating the oscillatory element with an energy source inenergetic contact with said oscillatory element so as to inducemicrostreaming in said fluid. The energy is optionally acoustic energyand the energy source is optionally an acoustic source. Any devicedescribed herein and equivalents are operable to induce microstreamingin a fluid.

Microstreaming in a fluid directionally oriented is capable of inducinga pumping action in the fluid so as to move fluid through a fluidpassage. As such, processes of pumping a fluid in a fluid passage arealso provided. Processes include contacting a fluid in a fluid passagewith an oscillatory element, the oscillatory element or oscillatingportion thereof oriented relative to an the longitudinal direction ofthe fluid passage by a tilting angle from 0 degrees to 180 degrees,optionally 0 to 70 degrees, where 0 degrees is parallel to thelongitudinal direction of the fluid passage at the location of theoscillatory element; and oscillating the oscillatory element with anenergy source in energetic contact with the oscillatory element so as toinduce directional microstreaming in the fluid. The energy is optionallyacoustic energy and the energy source is optionally an acoustic source.Any device described herein and equivalents are operable to inducemicrostreaming in a fluid.

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.

EXAMPLES Example 1 Sharp Edge Based Oscillatory Elements in a Micromixer

An exemplary single passage apparatus is illustrated in FIG. 1A. Asingle-layer fluid passage 12 is formed from polydimethylsiloxane (PDMS)with eight oscillatory elements illustrated as a sharp edge 14 on itssidewalls 16 (four edges on each side). The single-layer PDMSmicrochannel was fabricated using soft lithography and the mold replicatechnique. A silicon mold for the microchannel was patterned inphotoresist (Shipley 1827, MicroChem, Newton, Mass.) and etched withDeep Reactive Ion Etching (DRIE, Adixen, Hingham, Mass.). The mold wasthen coated with 1H,1H,2H,2H-perfluorooctyl-trichlorosilane (SigmaAldrich, St. Louis, Mo.) to reduce its surface energy and any subsequentdamage to the PDMS channel during the demolding process. Sylgard™ 184Silicone Elastomer Base and Sylgard™ 184 Silicone Elastomer Curing Agent(Dow Corning, Midland, Mich.) were mixed at a 10:1 weight ratio and castonto the silicon mold. The uncured PDMS on the silicon mold was thendegassed in a vacuum chamber for 2 h to remove any air bubbles and latercured at 65° C. for 45 min. After removing the cured PDMS from the mold,the inlets and the outlets were drilled into the PDMS using a siliconcarbide drill bit (model 220/395, Dremel). The microfluidic flow channelwas then bonded to a micro cover glass used as a substrate 18 that hadbeen pre-treated with oxygen plasma. An acoustic source illustrated as apiezoelectric transducer 20 (model no. 273-073, RadioShack Corp.) isthen attached adjacent to the fluid channel 12 by using epoxy(PermaPoxy™ 5 Minute General Purpose, Permatex).

To demonstrate and characterize the fluid flow pattern inside the aboveexemplary fluid passage due to the acoustic streaming, a solutioncontaining 1.9 μm diameter DRAGON GREEN fluorescent beads (BangsLaboratory) was first infused into the fluid passage. FIG. 2A shows theflow pattern of fluorescent beads in the absence of acoustic activation(with the piezoelectric transducer OFF). In the presence of acousticactivation (with the piezoelectric transducer ON), oscillatingsharp-edges induced a strong acoustic microstreaming effect (FIG. 2B).The microstreaming greatly enhanced the mass transport of the two fluidsby perturbing the bulk flow and breaking the interface of laminar flow,thereby enabling fast and homogeneous mixing.

The mixing performance of the exemplary sharp-edge-based micromixer wascharacterized by injecting DI water and fluorescent dye (fluorescein)into the fluid passage through two separate inlets (24, 26 in FIG. 1A).The sharp-edges 14 were acoustically oscillated by the piezoelectrictransducer 18 that was driven by an amplified sine-wave signal from afunction generator and an amplifier. To determine the frequency at whichthe oscillating sharp-edges generate the strongest acoustic streamingeffect, the device was tested with 15° tip angle sharp-edges, and sweptthe frequency with a 50 Hz increment from 1 kHz to 100 kHz. Experimentalresults indicated that the strongest acoustic streaming effect wasgenerated when the sharp-edges were excited at the frequency of 4.50kHz. FIG. 3A shows the unmixed laminar flow profile at a flow rate of 1μl/min with the piezoelectric transducer OFF, in which a clear fluidinterface was observed. FIGS. 3B-3D show the mixing results due to thepresence of acoustic waves at frequencies of 4.25 kHz, 4.50 kHz, and4.75 kHz, respectively. Homogeneous mixing of DI water and fluoresceinwas achieved when the sharp-edges were excited at frequencies of 4.50kHz and 4.75 kHz, while incomplete mixing was observed at a frequency of4.25 kHz. To further verify the mixing performance and identify theoptimized driving frequency of the piezoelectric transducer, thecross-sectional dye concentration profiles (the dashed lines in FIG.3A-D) were plotted by measuring the grey scale value of the experimentalimages. FIG. 3E shows the normalized dye concentration profile acrossthe flow passage width for the three driving frequencies. Theconcentration profiles show that a uniform gray-scale value distributionacross the channel width was observed at a frequency of 4.50 kHz,suggesting that 4.50 kHz is the proper driving frequency to develop thestrongest acoustic streaming phenomenon and achieve optimized mixingperformance for this apparatus at this flow rate. As a result, thefrequency of 4.50 kHz was used in all the following experiments withthis apparatus. It will be understood by those of skill in the art thatother driving frequencies may be used in other configurations and/or forother fluids, flow rates, etc.

Once the driving frequency was determined, the effect of the tip angleof sharp-edges on the mixing performance was investigated. Toquantitatively characterize the mixing performance along the entirelength of the flow passage, the mixing index (M) of fluids was measuredat five different positions (indicated as 1, 2, 3, 4, and 5 in FIG. 4A)along the channel. The mixing index is defined as the standard deviationof normalized gray-scale values, which were extracted from theexperimental images obtained. A mixing index of 0.5 indicates completelyunmixed fluids, while a mixing index of 0.0 indicates completely mixedfluids. A mixing index of 0.1 was chosen as the upper-level thresholdfor acceptable mixing. FIG. 4A-D shows the mixing efficiencies of thefour different tip angles of sharp-edges at a flow rate of 2 μl/min (4μl/min for the total flow rate of the two co-injected fluids), a drivingfrequency of 4.50 kHz, and a driving voltage of 31 V (peak to peak).With a tip angle of 15° a mixing index of 0.065 was achieved at position2, suggesting excellent mixing of DI water and fluorescein. Forsharp-edges with a tip angle of 30°, complete mixing was observed atposition 5 suggesting that a longer mixing distance was required.Incomplete mixing was observed for sharp-edges with a tip angle of 45°(FIG. 4D). With a tip angle of 60°, a side-by-side laminar flow wasobserved due to the unmixed fluids (even in the presence of acousticwave), and only negligible mixing, which was caused by diffusion, wasobserved at downstream positions (FIG. 4E). The results showed that asthe tip angle of sharp-edges decreased, the mixing performancesignificantly improved. The results can be explained by approximatingthe oscillation of sharp-edges as the cantilever vibration. Forcantilever vibration, one can use the following equation,

k=F/δ=Ewt ³/4L ³  (1)

where k is the spring constant, E is the Young's modulus of material, wis the width of cantilever, t is the thickness of the cantilever, and Lis the length of the cantilever. Sharp-edges with different tip anglesin this study all have the same values for Young's modulus, equivalentwidths (50 μm), and equivalent lengths (250 μm). The only variable thatchanges with varying tip angles is the thickness, which increases as theangle increases. Thus sharp-edges with a smaller tip angle should have alower spring constant. If the input power is constant, a lower springconstant of the cantilever will cause a larger vibration amplitude atthe free end of the cantilever. Treating each single sharp-edge as onecantilever, similarly, the sharp-edges with a tip angle of 15° shouldhave the largest vibration amplitude because of its smallest springconstant. This explains why the sharp-edges with the tip angle of 15°induced stronger acoustic streaming effects than those with tip anglesof 30°, 45°, or 60°.

In light of the above, tip angles of less than 30 degrees are preferredfor some embodiments, while greater angles may be acceptable for otherembodiments. For example, tip angles of 45 degrees or less may be usedin some embodiments.

Mixing performance was further characterized by applying differentdriving voltages to the piezoelectric transducer. FIG. 5 shows themixing performance with different driving voltages at a flow rate of 2μl/min and a driving frequency of 4.50 kHz. The results show that as thedriving voltage of the piezoelectric transducer increased, the mixingefficiency was improved, and acceptable mixing was observed startingfrom position 2 with driving voltages of 23 VPP, 31 VPP, and 39 VPP.With a driving voltage of 15 VPP, the acceptable mixing index wasachieved at position 3, suggesting that a lower driving voltage inducedweaker acoustic streaming effects; therefore a longer mixing distancewas required.

FIG. 6A-D shows the mixing efficiency at different flow rates (1, 2, 3,4, and 5 μl/min) with a driving frequency of 4.50 kHz and a drivingvoltage of 31 VPP. At lower flow rates (1 and 2 μl/min), acceptablemixing was achieved at position 2, which suggests excellent mixing ofthe two fluids and shorter mixing distances were required for low flowrates (FIGS. 6A and 6B). For higher flow rates (3, 4, and 5 μl/min), themixing index at position 2 was increased with an increase in flow rate,and acceptable mixing was only observed after passing position 3. Theresults suggest that the mixing index increases as flow rate increases,since the ability to oscillate sharp-edges to induce acoustic streamingmight be suppressed by high flow rates. The upper limit of flow rate, bywhich a mixing index less than 0.1 was achieved after passing position 2(after the first pair of sharp-edges), was 2 μl/min (4 μl/min for thetotal flow rate of two coinjected fluids). Although mixing indices lessthan 0.1 were achieved with the flow rates higher than 2 μl/min, theywere only observed after passing position 3, suggesting a longer mixingdistance.

The mixing time of the sharp-edge-based micromixer was alsocharacterized. The average mixing time (Ts) was estimated using thefollowing equation,

τ_(s)=L_(mix)/V_(avg)  (2)

where τ_(s) is the mixing time, L_(mix) is the distance from unmixed tocompletely mixed regions, and V_(avg) is the average fluid velocity. Themixing distance was measured to be approximately 400 μm from FIG. 6B,and the average fluid velocity was calculated to be 2.2 mm/s by dividingthe combined flow rate by the cross-sectional area of the channel (600μm by 50 μm). The mixing time was thus calculated to be around 180 mswhich is comparable to those of existing microfluidicmixers.^(42-45,51,53) The inventors believe that the mixing time can befurther shortened through the optimization of design parameters, such asthe distance between consecutive single sharp-edge or the height ofsharp-edges.

In conclusion, the present invention provides an acoustofluidicmicromixer based on the acoustic streaming effects induced byoscillating sharp-edges. The recirculating flows induced by theoscillation of sharp-edges allow two fluids to interchange and thusenhances the mass transport across the channel, greatly improving themixing efficiency. Experiments demonstrate that homogeneous mixingacross the channel width can be achieved and the mixing time wascalculated to be ˜180 ms. The effects of the sharp-edge geometry, thedriving frequency, the driving voltage, and the flow rates on mixingperformance were investigated. The sharp-edge-based acoustofluidicmicromixer has many desirable characteristics, such as its excellentmixing performance, simplicity, convenient and stable operation, fastmixing speed, and ability to be toggled on-and-off. Thesecharacteristics make it promising for a wide variety of lab-on-a-chipapplications.

Example 2 Cellular Calcium Responses Measured with an AcustofluidicMicromixer

The apparatus of Example 1 was used to probe the calcium responses ofU251 cells. Briefly, stimulus and buffer, were co-injected through twoseparate inlets, and they could be uniformly mixed due to the acousticstreaming effects that controlled by the actuation of PZT. The fluidpassage was divided into two regions of interest (ROI) along the fluidicinterface, ROI 1 and ROI 2 as illustrated in FIG. 7A. When the PZT wasinactivated, two ROIs were only filled with the stimulus and the buffer,respectively, while being filled with uniformly mixed solution, once thePZT was activated as shown in FIG. 7B and C, respectively. By alternateactuation of the PZT, temporal control over mixing is achievedgenerating different chemical signal profiles at two the ROIs asillustrated in FIG. 7D. This enables the study of cellular responses tostimuli with two different concentration amplitudes. Adjusting the burstfrequency and the duty cycle of input signals of PZT allows generationof various pulsed chemical signal profiles using our mixing-based cellstimulator as illustrated in FIG. 8 with various on/off time periods.

The calcium response of U251 cells exposed to sustained and pulsedstimulations of ionomycin, a calcium ionophore was studied. Briefly,U251 cells were first loaded into the fluid passage, and randomly seededin the two ROIs. After seeding, HBSS containing 2 μM Fluo4-AM was gentlyand slowly delivered into the channel to stain the cells, followed bywashing with phosphate buffered saline (PBS). Then medium (PBS) andmedium containing 2 μM ionomycin were injected into separate inputs andallowed to flow together through the fluid passage. U251 cells in ROI 1and ROI 2 were subjected to differing concentration amplitudes ofionomycin between 2 to 1 μM and between 0 and 1 μM, respectively. Withexposure to a sustained stimulus of 60 sec, U251 cells in ROI 1 showed adecayed fluorescence intensity over time, after that, they expressed anincreased fluorescence intensity [FIG. 7A]. By contrast, those in ROI 2showed enhanced florescence intensity over time [FIG. 7B]. The cellsresponded to the pulsed stimulus at a frequency of 0.0625 Hz. Whenexposed to a pulsed stimulus at higher burst frequency of signals,including 0.25 Hz and 1 Hz, U251 cells in both ROIs failed to respondfaithfully to the oscillating concentrations of stimulus, rather,integrated the signal. The results suggest that the cells are actuallyacting as a low-pass filter.

Example 3 Production of Fine Gradient Profiles in an AcustofluidicMicromixer

An acoustofluidic micromixer is formed substantially as in Example 1with a wide fluid passage including oscillatory elements in the form ofsharp edges dispersed transversely from substrate to passage edge suchthat fluid is capable of flowing around both adjacent surfaces from baseto tip. Two illustrative embodiments tested are presented in FIG. 9Aillustrating a linear pattern and FIG. 9B illustrating a parabolicpattern of the oscillatory elements.

Fluorescein and PBS solutions are transferred into the fluid channel tocharacterize chemical gradients produced by the apparatuses. Upon theactuation of the piezoelectric transducer, the sharp edges areacoustically oscillated to generate acoustic streaming effects aroundthe tip of each sharp-edge, and to mix the fluids from the first inletcontaining a fluorescein composition and a second inlet containingwater. Due to the step-like arrangement of sharp-edge oscillatoryelements, the fluids are mixed in a step-wise fashion producing a finegradient profile from one edge of the fluid passage to the opposingedge. A linearly varying gradient is generated by the arrangement ofoscillatory elements of FIG. 9A. A parabolic chemical gradient isgenerated by the arrangement of oscillatory elements in FIG. 9Bemploying two water inlets and a central fluorescein containing inlet.

As illustrated in FIG. 10A, using the apparatus of FIG. 9A, when thepiezoelectric transducer was OFF, a side-by-side laminar flow wasobserved due to the nature of low Reynolds number in microfludicchannel. Once the piezoelectric transducer was actuated, acousticmicrostreaming induced mixing in a step-wise fashion was observed,thereby generating concentration gradients of fluorescein as illustratedin FIGS. 10B-D. To verify the gradients generated, the cross-sectionalfluorescence intensity profiles at downstream position ROI (dashed linein FIG. 9) were plotted as illustrated in FIG. 10E. As expected, lineargradients were obtained. In addition, gradients can be tuned as uponaltering the driving voltage of the piezoelectric transducer withincreasing voltage producing gradients with finer profiles.

FIG. 11A-D illustrates shows the concentration gradients that generatedusing the design in FIG. 9B. Different concentration gradients wereexperimentally observed when different driving voltages of thepiezoelectric transducer were applied FIG. 11B-D. As illustrated in FIG.11E, varying the voltage of the piezoelectric transducer resulted infiner gradient profiles.

Generation of gradients with more complex profiles is possible bychanging the driving voltages or the arrangement of oscillatoryelements. In addition, temporally changing gradients can be generated byeasily controlling the driving voltage and the actuation time of thepiezoelectric transducer.

Example 4 An Acustofluidic Micropump

An acustofluidic micropump is formed substantially as described inExample 1 with a plurality of oscillatory elements arranged in a fluidpassage at a tilting angle of less than 90 degrees causing a tilting ofthe oscillatory elements in the same direction. FIG. 12A schematicallyshows the design and working mechanism of the acoustofluidic micropumpaccording to this exemplary design. Briefly, the acoustofluidicmicropump was made by bonding a single-layer PDMS fluid passage formedby the methods of Example 1 with a piezoelectric transducer (Part no.81-7BB-27-4L0, Murata Electronics) attached adjacent to it onto a singleglass slide using a thin layer of epoxy (PermaPoxy™ 5 Minute GeneralPurpose, Permatex).

To demonstrate pumping behaviour, the flow channel was designed to be arectangular recirculating channel composed of four interconnectedchannels—left-channel, right-channel, upper-channel and lower-channel.The lower channel includes the pumping region and was designed withtwenty tilted oscillatory elements on its sidewalls (ten on each side),while all the other three channels were straight channels without anystructure. The piezoelectric transducer, activated by amplifiedsine-wave signals from a function generator (AFG3011C, Tektronix) and anamplifier (25A250A, Amplifier Research), was used to acousticallyoscillate the oscillatory elements to generate acoustic streamingeffects. As illustrated in FIG. 12B, when acoustically oscillated by theactivation of the piezoelectric transducer, the tilted oscillatoryelements generate a directional microstreaming pattern around the tip,thereby producing a net force pointing towards the direction thatoscillatory element is orientated. Fluid pumping occurs as a result ofthe net forces pushing the bulk fluid to flow forward. FIG. 12Cillustrates the dimensions of the acoustofluidic micropump: themicrochannel has a width of 600 μm and a depth of 100 μm, and eachsharp-edge structure is identical. Different tilting angles (β) ofoscillatory elements, including 30°, 45°, 60° and 70°, were chosen toinvestigate the resulting pumping behaviour and determine optimal anglefor best pumping performance.

As shown in FIG. 13A, the recirculating fluid passage was designed toproduce fluid pumping in a counter-clockwise direction employing 30°tilted oscillatory elements, as shown by the inset. A solutioncontaining DI water and 1.9 μm diameter DRAGON GREEN fluorescent beads(Bangs Laboratory) was injected into the channel to characterize theacoustic streaming patterns induced by the oscillation of tiltedoscillatory elements. The acoustic frequency was swept from 1 kHz to 100kHz 50 Hz increments. The microstreaming patterns, as shown in FIG. 13B,were developed around the tips of oscillating oscillatory elements inthe pumping region, when the piezoelectric transducer was activated at6.5 kHz, the frequency at which the pumping occurs on account of theinduced acoustic streaming effect. For an acoustic micropump of thisexemplary design, 6.5 kHz was the working frequency for thepiezoelectric transducer to activate the pumping action, and thus thisfrequency was used for all the following experiments.

To further investigate the influence of the tilting angle of oscillatoryelements on the pumping performance, a mixture of DI water mixed withpolystyrene beads of different diameters (20 μm and 0.9 μm) was injectedinto the channel. And the estimated average flow rate inside the channelwas calculated by tracking average bead velocity in the upper channel,in which 50-100 beads were randomly selected and tracked for eachindependent experiment. In addition to the effect of tilting angle, thepumping performance under different input voltages of the piezoelectrictransducer was also characterized. FIG. 14A illustrates the pumpingperformance of the four different tilting angles of oscillatory elementsunder different input voltages. The results show that when thepiezoelectric transducer was activated with voltages ranging from 5 Vppto 50 Vpp, pumping occurred at all tilting angles. As the tilting anglewas decreased, the generated pumping flow rate increased. Of the fourdifferent tilting angles, the device with 30° tilted sharp-edgestructures, as shown in FIG. 14A, generated significantly greaterpumping flow rate, and with a voltage of 50 Vpp, it generated a flowrate as high as 8 μL/min, which corresponds to a calculated pumpingpressure of 76 Pa. Lower pumping flow rates generated by 45°, 60° and70° tilted oscillatory elements can be attributed to the fact that withlarger the tilting angle, the generated net force points more towardsthe direction perpendicular to the flow direction, thereby weakening theforce that could push the bulk fluid to flow forward. In addition, asthe voltage was increased, the pumping flow rate also increased,indicating that the pumping flow rate could be controlled by adjustingthe input voltages. Using the tilted oscillatory elements in the fluidchannel, wide-range pumping flow rates, from nL/min to μL/min, weregenerated by adjusting the input voltage to the piezoelectrictransducer. Aside from the function of continuous fluid pumping, FIG.14B demonstrates that alternately switching the piezoelectric transducerto ON and OFF at various burst frequencies, 0.5 Hz (T=2 sec) and 2 Hz(T=0.5 sec), that the function of pulsatile fluid pumping could berealized, thereby indicating that the profile of the pumping flow ratecould be modulated by programming the input signal to the piezoelectrictransducer.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

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Patents, publications, and applications mentioned in the specificationare indicative of the levels of those skilled in the art to which theinvention pertains. These patents, publications, and applications areincorporated herein by reference to the same extent as if eachindividual patent, publication, or application was specifically andindividually incorporated herein by reference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. An acoustofluidic apparatus comprising: a fluid passage, said fluid passage comprising at least one sidewall, at least one inlet, and at least one outlet; an oscillatory element comprising a tip extending into said fluid passage; and an acoustic source in acoustic contact with said oscillatory element, said acoustic source operable to vibrate said oscillatory element or a portion thereof, so as to create microstreaming in a fluid within said fluid passage.
 2. The acoustofluidic apparatus of claim 1, said oscillatory element comprising two adjacent surfaces extending between said sidewall and said tip, said adjacent surfaces being disposed at a tip angle with respect to each other, the tip angle being less than 180 degrees.
 3. The acoustofluidic apparatus of claim 2, wherein the tip angle is less than or equal to 45 degrees.
 4. The acoustofluidic apparatus of claim 1, said tip having a surface with a width and a length.
 5. The acoustofluidic apparatus of claim 4, said length equal to the height of said fluid channel.
 6. The acoustofluidic apparatus of claim 4, said width is from 5 to 100 micrometers.
 7. The acoustofluidic apparatus of claim 1, said oscillatory element extending at a tilting angle relative to the length of said sidewall, said tilting angle being 90 degrees or less.
 8. The acoustofluidic apparatus of claim 7, said tilting angle is from 0 degrees to 70 degrees.
 9. The acoustofluidic apparatus of claim 1, wherein the acoustic source is a piezoelectric transducer.
 10. The acoustofluidic apparatus of claim 1, wherein the acoustic source is operable to vibrate the oscillatory element at a frequency in the range of 2 Hz to 900 MHz.
 11. A process of inducing microstreaming in a fluid comprising: contacting a first fluid in a fluid passage with a oscillatory element; and acoustically oscillating said oscillatory element with an acoustic source in acoustic contact with said oscillatory element so as to induce microstreaming in said first fluid.
 12. The process of claim 11 wherein said oscillatory element comprises a tip angle of 45 degrees or less.
 13. The process of claim 11 wherein said oscillatory element is oriented relative to an edge of said fluid passage by a tilting angle from 0 degrees to 70 degrees.
 14. The process of claim 11 wherein said oscillatory element oscillates at a frequency in the range of 2 Hz to 900 MHz.
 15. The process of claim 11 wherein said microstreaming induces mixing of said first fluid and a second fluid, and optionally a third fluid, within said fluid channel.
 16. The process of claim 15 producing a concentration gradient of a component of said first fluid, said second fluid, optionally said third fluid, or any combination thereof.
 17. The process of claim 15 further comprising contacting said first fluid and said second fluid with a plurality of oscillatory elements off set and generating a gradient profile between said first fluid and said second fluid, and optionally said third fluid.
 18. The process of claim 15 further comprising alternating said acoustic source from an on state to an off state to produce a temporal gradient waveform of concentration of said first fluid and said second fluid.
 19. A process of pumping fluid through a channel comprising: contacting a fluid in a fluid passage with an oscillatory element, said oscillatory element, or oscillating portion thereof, oriented relative to an edge of said fluid passage by a tilting angle from 0 degrees to 180 degrees; and acoustically oscillating said oscillatory element with an acoustic source in acoustic contact with said oscillatory element so as to induce directional microstreaming in said fluid.
 20. The process of claim 19 wherein said tilting angle is from 0 degrees to 70 degrees. 